The major clinical feature associated with poor prognosis and survival in cancer patients remains the development of metastasis. Evidence indicates that MT 1-MMP, a membrane-anchored matrix metalloproteinase (MMP), plays a key role in the process of tumor metastasis and angiogenesis. MT 1-MMP is a maj or mediator of pericellular proteolysis by promoting extracellular matrix degradation (ECM) and initiating the cell surface activation of MMP-2 (gelatinase A), another key enzyme for tumor metastasis. The long-term goal of this application is to develop novel approaches to specifically inhibit the activity of MT 1-MMP and gelatinases in cancer tissues. As a membrane-tethered enzyme, MT 1-MMP undergoes autocatalytic processing and ectodomain shedding, two processes that control the amount of active enzyme on the cell surface. Preliminary evidence shows that reversible synthetic MMP inhibitors inhibit MT 1-MMP processing and shedding resulting in the accumulation of active enzyme on the cell surface. Paradoxically, reversible MMP inhibitors enhance MMP-2 activation by MT1-MMP in the presence of TIMP-2. Enzyme inhibition kinetic data support a model in which TIMP-2 displaces the reversible synthetic MMP inhibitor from the active site of MT 1-MMP facilitating MMP-2 binding and activation. These observations represent a paradigm in the regulation of the MT 1-MMP/gelatinase axis by reversible synthetic MMP inhibitors and highlight the limitations of current approaches for MMP inhibition. A novel strategy for MMP inhibition involving mechanism-based inhibitors developed in our labs has recently produced the first prototype irreversible MMP inhibitor for MMP-2 and MMP-9. Here we propose to pursue this approach for inhibition of MT 1-MMP activity using a comprehensive and multidisciplinary chemical, biochemical and biological approach. Specifically, we will (1) produce mechanism-based irreversible inhibitors for MT1-MMP and gelatinases, (2) characterize the mechanism-based inhibitors to establish inhibition and specificity parameters and to assess their metabolic fate in vitro, (3) investigate the effects of mechanism-based inhibitors on MT1-MMP/gelatinase activity in cells to define their role in MT 1-MMP processing, shedding, pro-MMP-2 activation and interactions with TIMPs and (4) establish the effectiveness of mechanism-based inhibitors on (ECM) degradation and invasion. It is expected that the results would create a new paradigm in regulation of MMPs by synthetic MMP inhibitors, opening previously unexplored avenues for targeting MMPs in prevention of both tumor growth and metastasis.